Abstract
Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11Δ to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements.
Copyright © 2013 Elsevier Inc. All rights reserved.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Blotting, Western
-
DNA Breaks, Double-Stranded
-
DNA Helicases / genetics
-
DNA Helicases / metabolism*
-
DNA Repair
-
DNA, Fungal / chemistry
-
DNA, Fungal / genetics
-
DNA, Fungal / metabolism*
-
DNA, Single-Stranded / chemistry
-
DNA, Single-Stranded / genetics
-
DNA, Single-Stranded / metabolism
-
DNA-Directed DNA Polymerase / genetics
-
DNA-Directed DNA Polymerase / metabolism
-
Endodeoxyribonucleases / genetics
-
Endodeoxyribonucleases / metabolism
-
Exodeoxyribonucleases / genetics
-
Exodeoxyribonucleases / metabolism*
-
Luminescent Proteins / genetics
-
Luminescent Proteins / metabolism
-
Microscopy, Fluorescence
-
Models, Genetic
-
Mutation
-
Nucleic Acid Conformation
-
Protein Binding
-
Rad51 Recombinase / genetics
-
Rad51 Recombinase / metabolism
-
RecQ Helicases / genetics
-
RecQ Helicases / metabolism*
-
Replication Protein A / genetics
-
Replication Protein A / metabolism*
-
Saccharomyces cerevisiae / genetics
-
Saccharomyces cerevisiae / metabolism
-
Saccharomyces cerevisiae Proteins / genetics
-
Saccharomyces cerevisiae Proteins / metabolism*
Substances
-
DNA, Fungal
-
DNA, Single-Stranded
-
Luminescent Proteins
-
Pol32 protein, S cerevisiae
-
RFA1 protein, S cerevisiae
-
Replication Protein A
-
Saccharomyces cerevisiae Proteins
-
RAD51 protein, S cerevisiae
-
Rad51 Recombinase
-
DNA-Directed DNA Polymerase
-
Endodeoxyribonucleases
-
Exodeoxyribonucleases
-
MRE11 protein, S cerevisiae
-
exodeoxyribonuclease I
-
SGS1 protein, S cerevisiae
-
DNA Helicases
-
DNA2 protein, S cerevisiae
-
RecQ Helicases